Biosynthetic regulatory network of flavonoid metabolites in stems and leaves of Salvia miltiorrhiza

Abstract

Flavonoid secondary metabolites can treat and prevent many diseases, but systematic studies on regulation of the biosynthesis of such metabolites in aboveground parts of Salvia miltiorrhiza are lacking. In this study, metabonomic and transcriptomic analyses of different S. miltiorrhiza phenotypes were conducted to explore pathways of synthesis, catalysis, accumulation, and transport of the main flavonoid secondary metabolites regulating pigment accumulation. Tissue localization and quantitative analysis of flavonoid secondary metabolites were conducted by laser scanning confocal microscopy (LSCM). A total 3090 differentially expressed genes were obtained from 114,431 full-length unigenes in purple and green phenotypes, and 108 functional genes were involved in flavonoid biosynthesis. Five key phenylpropane structural genes (PAL, 4CL, ANS, 3AT, HCT) were highly differentially expressed, and four transcription factor genes (MYB, WRKY, bHLH, bZiP) were identified. In addition, six GST genes, nine ABC transporters, 22 MATE genes, and three SNARE genes were detected with key roles in flavonoid transport. According to LSCM, flavonoids were mainly distributed in epidermis, cortex, and collenchyma. Thus, comprehensive and systematic analyses were used to determine biosynthesis, accumulation, and transport of flavonoids in stems and leaves of different S. miltiorrhiza phenotypes. The findings will provide a reference for flavonoid production and cultivar selection.

Figure 1

Response of S. miltiorrhiza Phenotypes to flavonoids. (A) Surface view of green and purple stem internode. (B) Surface view of green leaf and the leaves with purple edges. (C) Periderm tissue of green and purple stems under fluorescence microscope. (D) Periderm and mesophyll tissue of green leaves and the leaves with purple edges under fluorescence microscope. (E) A model to explain the internal mechanism of purple pigmentation in stems and leaves of S. miltiorrhiza. (F) The main secondary metabolic pathway in S. miltiorrhiza. (G) DAMs in different phenotypes of S. miltiorrhiza stems and leaves (L1: green leaf, L2: the leaves with purple edges; S1: green stem, S2: purple stem, pos indicated positive ion mode; neg indicated negative ion mode).

Figure 2

(A) GO functional classification of differentially expressed genes in L1 (green leaf), and L2 (the leaves with purple edges) in S. miltiorrhiza. (B) GO functional classification of differentially expressed genes in S1 (green stem), and S2 (purple stem). (C) Scatter plots of the KEGG pathway enrichment statistics in L1–L2. (D) Scatter plots of the KEGG pathway enrichment statistics in S1–S2.

Figure 3

(A) Expression patterns of flavonoids biosynthetic genes in stems and leaves samples of S. miltiorrhiza. (B) Expression of flavonoids biosynthetic transcription factors in nine samples of S. miltiorrhiza. The horizontal axis represents log2 (FPKM + 1) of the sample and the vertical axis represents genes. L1(green leaf), L2(the leaves with purple edges), S1(green stem), S2 (purple stem).

Figure 4

(A) Metabolite classification bar chart in BGI library. (B) Bar chart of metabolite KEGG function annotation. (C) Bar chart of metabolite classification.

Figure 5

(A) Statistical map of differential metabolites (DAMs). (B) The Hierarchical cluster analysis of differential accumulated metabolites (DAMs) in L1–L2, S1–S2 of S. miltiorrhiza. (C) Bubble Diagram of metabolic pathway enrichment analysis in L1–L2.

Figure 6

Molecular regulation of color phenotype characters of S. miltiorrhiza. (A) A working model to explain the internal mechanism of purple pigmentation in the aboveground part of S. miltiorrhiza. (B) Schematic diagram of flavonoids biosynthesis and regulation in S. miltiorrhiza. The redder the color of the color block, the higher the expression, and the bluer the color, the lower the expression.

Figure 7

Expression levels of 10 genes involved in the flavonoids biosynthetic pathway by qRT-PCR analysis.

Figure 8

Leaves and stems were stained with NA solution and observed with CLSM (50 µm). The figure showed the distribution of flavonoids in the cross section of S. miltiorrhiza after staining. (A,B,E,F) indicated the distribution of flavonoids in the AF488 channel. (C,D,G,H) Indicated the distribution of flavonoids in superimposed bright field of AF488 channel. (A,C) Indicated the purple stem. (B,D) Indicated the green stem; (E,G) indicated the leaf with purple edge. (F,H) Indicated the green leaf.